The Mechanism Of Interferon-stimulated Gene 12(ISG12) On Inhibition Of Newcastle Disease Virus Replication

Newcastle disease(ND)is one of the most serious avian infectious diseases caused by Newcastle disease virus(NDV).It has the characteristics of high fever,difficulty in breathing,contact sexual transmission,and septicemia.The poultry industry in China and the world has caused serious economic losses due to ND.At present,immunization is the main way to prevent and control Newcastle disease,but does not achieve the desired results in some cases,so it is important to explore new prevention strategies.In the early stage of the laboratory,RNA-seq technology was used to screen out multiple differentially expressed genes related to immunity after NDV infectionincluding interferon-stimulated genes 12(1)and 12(2)(ISG12(1)/(2)).This study focused on mechanism of chicken cells via ISG12(1)/(2)to block NDV replication.The main findings of this paper are as follows:(1)Both ISG12(1)and ISG12(2)were significantly up-regulated after NDV infection.10-day-old SPF chicken embryos and 4-week-old SPF chickens infected with NDV F48E9,both transcriptome sequencing and RT-qPCR showed significant upregulation of ISG12(1)/(2).ISG12(1)was up-regulated after infection of CEFs and DF-1 cells with NDV strains of varying virulence.In addition,the expression of ISG12(2)was also up-regulated in DF-1 infected with NDV F48E9.It suggests that ISG12 may play a role in NDV infection.(2)Both ISG12(1)and ISG12(2)inhibited replication of NDV.The eukaryotic expression plasmid of chicken ISG12(1)/(2)was constructed and transfected into DF-1 cells to detect the effect of overexpression of ISG12(1)/(2)on NDV infection.Overexpression of ISG12(1)/(2)can significantly inhibit viral proliferation and reduce the virus titer in cell supernatants.When the interfering RNA(siRNA)of ISG12(1)/(2)was transfected into DF-1cells,the expression of ISG12(1)/(2)was inhibited,which promoted NDV proliferation.(3)ISG12(1)promotes cell apoptosis via mitochondrial-dependent pathway and so as to hinder Newcastle disease virus replication.ISG12(1)was overexpressed/knockdown in DF-1 cells,and apoptosis was detected by flow cytometry.The results showed that ISG12(1)promoted apoptosis.ISG12(1)was localized to mitochondria confirmed by laser confocal microscopy,and ISG12(1)also colocalized with the pro-apoptotic protein Bax and promoted the redistribution of Bax from cytoplasm to mitochondria.The protein and mRNA levels of mitochondrial apoptosis pathway-related molecules were detected by Western blot andRT-qPCR,respectively.The results showed that after overexpression of ISG12(1),proapoptotic genes Bax,Bak,Cyt c,caspase-3 and caspase-9 was significantly up-regulated,and the anti-apoptotic genes Bcl-2 and Bcl-xl were significantly down-regulated,and knocking down ISG12(1)gave the opposite result.This indicates that ISG12(1)inhibits NDV replication by mitochondria-mediated apoptosis in NDV infection.(4)ISG12(2)is resistant to NDV infection by activating the type I interferon signaling pathway.First,the expression of apoptosis-related genes was detected by Western blot after overexpression/knockdown of ISG12(2).The results showed that ISG12(2)did not cause changes in apoptosis-related genes.Furthermore,flow cytometry experiments also showed that the effect on apoptosis was not significant when overexpressing ISG12(2).The above indicates that ISG12(2)exerts a non-apoptotic antiviral mechanism.After overexpression of ISG12(2),RT-qPCR was used to detect IFNs-related cytokine changes.ISG12(2)significantly promoted the expression of IFNa and IFN?,and also promoted MDA5,MAVS,IRF7 and NF-?B expression.Conversely,overexpression of IFN?,IFN? also promoted the expression of ISG12(2).In addition,cell supernatant overexpressing ISG12(2)inhibited the proliferation of porcine vesicular stomatitis virus(VSV).The opposite result was obtained by knocking down the expression of ISG12(2).It is indicated that ISG12(2)can regulate the production of interferon and play an anti-NDV role.14 host proteins interacting with ISG12(2)were screened by mass spectrometry.Through Go classification and pathway analysis,these proteins may be involved in biological processes such as biosynthesis,metabolism,and participate in Toll-like receptor signaling pathways,Jak-STAT signaling pathways,etc.This provides a basis for further study of antiviral mechanism of the ISG12(2).In summary,ISG12(1)and ISG12(2)were significantly up-regulated after NDV infection,and ISG12(1)induced apoptosis and inhibited NDV replication by activating the mitochondrial apoptotic pathway and colocalizing with the pro-apoptotic protein Bax.ISG12(2)was resistant to NDV infection by promoting the expression of interferon.Thus,these two members of the ISG12 protein family resist the invasion of pathogens through apoptosis and innate immunity,respectively,which ar the basic defense mechianisms of cell against viral infection.